Solid-phase proteoliposomes containing human immunodeficiency virus envelope glycoproteins

Abstract

The human immunodeficiency virus type 1 (HIV-1) exterior envelope glycoprotein gp120 mediates receptor binding and is the major target for neutralizing antibodies. A broadly neutralizing antibody response is likely to be a critical component of the immune response against HIV-1. Although antibodies against monomeric gp120 are readily elicited in immunized individuals, these antibodies are inefficient in neutralizing primary HIV-1 isolates. As a chronic pathogen, HIV-1 has evolved to avoid an optimal host response by a number of immune escape mechanisms. Monomeric gp120 that has dissociated from the functional trimer presents irrelevant epitopes that are not accessible on functional trimeric envelope glycoproteins. The resulting low level of antigenic cross-reactivity between monomeric gp120 and the functional spike may contribute to the inability of monomeric gp120 to elicit broadly neutralizing antibodies. Attempts to generate native, trimeric envelope glycoproteins as immunogens have been frustrated by both the lability of the gp120-gp41 interaction and the weak association between gp120 subunits. Here, we present solid-phase HIV-1 gp160DeltaCT (cytoplasmic tail-deleted) proteoliposomes (PLs) containing native, trimeric envelope glycoproteins in a physiologic membrane setting. We present data that indicate that the gp160DeltaCT glycoproteins on PLs are trimers and are recognized by several relevant conformational ligands in a manner similar to that for gp160DeltaCT oligomers expressed on the cell surface. The PLs represent a significant advance over present envelope glycoprotein formulations as candidate immunogens for HIV vaccine design and development.

FIG. 1.

Schematic diagram of the formation of PLs. (A) Beads conjugated with the 1D4 antibody recognizing the C9 tag were used to capture the C9-tagged gp160ΔCT glycoproteins from cell lysates. The beads were then washed extensively in detergent-containing buffer to remove non-C9-tagged proteins. (B) The beads were incubated with detergent-solubilized lipid and dialyzed. (C) During dialysis in PBS, the detergent is replaced by a lipid membrane which assembles around the gp160ΔCT glycoproteins.

FIG. 2.

Protein composition of PLs. (A) SDS-polyacrylamide gel of gp160ΔCT glycoproteins eluted from 5 × 10⁷ PLs, stained with Coomassie blue. Lane 1, 1 μg of affinity-purified gp160; lane 2, protein eluted from gp160ΔCT PLs; lane 3, protein eluted from beads conjugated with the 1D4 antibody. BSA, bovine serum albumin. (B) FACS analysis of PLs stained with the IgGb12 antibody (peak 2) and HIV-1 patient serum (peak 3), compared to anti-human FITC-conjugated secondary antibody alone (peak 1).

FIG. 3.

Size-exclusion chromatography and Western blotting of JR-FL gp160ΔCT glycoproteins eluted from Dynal beads under native conditions. 293T cells transiently expressing the JR-FL gp160ΔCT C9-tagged glycoproteins were lysed in CHAPS-containing buffer and incubated with Dynal beads conjugated with the 1D4 antibody. Beads were then washed and incubated in buffer containing 0.2 mM C9 peptide and 0.5 M MgCl₂ to elute the gp160ΔCT glycoprotein from the beads. (A) Approximately 5 μg of JR-FL gp160ΔCT glycoproteins was analyzed on a Superdex-200 gel filtration column. (B) Eluted fractions were collected, analyzed by SDS-PAGE under reducing conditions (2% BME and 100°C), and analyzed by Western blotting with a polyclonal anti-gp120 rabbit serum. (C) Eluted fractions were analyzed on an SDS-3 to 8% polyacrylamide gradient gel under nonreducing conditions (minus BME and 37°C) and under reducing conditions (2% BME and 100°C), respectively, and detected by Western blotting using a polyclonal anti-gp120 rabbit serum. Protein bands of apparent molecular masses consistent with trimeric gp160ΔCT glycoproteins (T), dimeric glycoproteins (D), and monomeric glycoproteins (M) are marked as indicated. FPLC, fast protein liquid chromatography.

FIG. 3.

Size-exclusion chromatography and Western blotting of JR-FL gp160ΔCT glycoproteins eluted from Dynal beads under native conditions. 293T cells transiently expressing the JR-FL gp160ΔCT C9-tagged glycoproteins were lysed in CHAPS-containing buffer and incubated with Dynal beads conjugated with the 1D4 antibody. Beads were then washed and incubated in buffer containing 0.2 mM C9 peptide and 0.5 M MgCl₂ to elute the gp160ΔCT glycoprotein from the beads. (A) Approximately 5 μg of JR-FL gp160ΔCT glycoproteins was analyzed on a Superdex-200 gel filtration column. (B) Eluted fractions were collected, analyzed by SDS-PAGE under reducing conditions (2% BME and 100°C), and analyzed by Western blotting with a polyclonal anti-gp120 rabbit serum. (C) Eluted fractions were analyzed on an SDS-3 to 8% polyacrylamide gradient gel under nonreducing conditions (minus BME and 37°C) and under reducing conditions (2% BME and 100°C), respectively, and detected by Western blotting using a polyclonal anti-gp120 rabbit serum. Protein bands of apparent molecular masses consistent with trimeric gp160ΔCT glycoproteins (T), dimeric glycoproteins (D), and monomeric glycoproteins (M) are marked as indicated. FPLC, fast protein liquid chromatography.

FIG. 4.

FACS analysis of the reconstituted PL membrane. (A) Occlusion of the 1D4 antibody by lipid membrane reconstitution. gp160ΔCT PLs with (peak 2) and without (peak 3) a reconstituted membrane were probed with anti-mouse Ig-PE antibody. Peak 1 shows staining with the same antibody of nonconjugated beads. (B) PLs with a reconstituted membrane containing 1% biotinylated lipid (peak 2) and beads without a reconstituted membrane (peak 1) were probed with avidin-FITC.

FIG. 5.

Fluorescence microscopic images of unconjugated beads (A) and PLs reconstituted with a membrane containing 1% DOPE-rhodamine (B).

FIG. 6.

(A) Binding of the gp41 antibody 2F5 to gp160ΔCT from HXBc2 (right) and JR-FL (left) on beads without a membrane (open squares) and fully reconstituted PLs (closed squares). PLs and beads were probed with increasing concentrations of 2F5 antibody and anti-human IgG-PE antibody, respectively, and analyzed by FACS. The mean fluorescence intensity (MFI) was plotted as percent maximal MFI at the given antibody concentration. (B) Binding of the antibodies 2G12 (left) and F105 (right) to the PLs (closed squares) and beads (open squares) was performed as described for 2F5 binding above.

FIG. 7.

Binding of a panel of anti-gp120 antibodies and sCD4 to YU2 gp160ΔCT glycoprotein expressed on 293T cells compared to that to YU2 gp160ΔCT glycoprotein on PLs. Cells (open circles) and PLs (closed squares) were incubated with increasing amounts of the indicated human antibodies, followed by detection with anti-human IgG-PE antibody. For detection of sCD4 binding to the glycoproteins, the rabbit anti-CD4 antibody T45 and anti-rabbit IgG-FITC were used. Following staining, samples were analyzed by FACS. The binding of ligands to gp160ΔCT PLs was plotted as percent normalized mean fluorescence intensity at serially diluted antibody concentrations. The percent normalized mean fluorescence intensity (MFI) values were calculated according to the formula [MFI − MFI (background)] × 100/[MFI (saturation) − MFI (background)]. Error bars indicate the range of values obtained for duplicate samples.

FIG. 8.

Induction of the 17b epitope by sCD4. Binding of the 17b antibody to gp160ΔCT glycoprotein on 293T cells and that on PLs were compared. Cells and PLs were incubated with (+) and without (−) sCD4 prior to binding of the 17b antibody and analyzed by FACS. Staining without preincubation with sCD4 was set as 100%, and increase of 17b binding is shown for 293T cells (open bars) and PLs (solid bars). MFI, mean fluorescence intensity.

FIG. 9.

Effect of proteolytic cleavage of HIV-1 envelope glycoprotein on ligand binding. Cleavage-competent YU2 gp160ΔCT glycoprotein (closed squares) and cleavage-defective YU2 gp160ΔCT glycoprotein (open circles) were expressed on 293T cells. Cells were incubated with increasing amounts of the indicated ligands followed by detection with anti-human IgG-PE. The anti-CD4 polyclonal rabbit antibody T45 and anti-rabbit IgG-FITC were used for the detection of sCD4 binding. Normalized mean fluorescence intensity was calculated as described for Fig. 7.